High speed sample supply device

ABSTRACT

Disclosed herein is a sample supply device that alternates between the supply of samples from one sample line while cleaning a second sample line and then supplying a second sample from the second sample line while cleaning the first sample line. This is repeated in rapid succession to allow greater speed in analyzing a plurality of samples in a shorter amount of time.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application60/781,103, entitled “High Speed Sample Supply Device” and filed on Mar.10, 2006, the contents of which are hereby incorporated by reference intheir entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a sample supply device for supplying aninspection instrument, such as a flow cytometer, with a plurality ofsamples at high speed.

2. Description of the Related Art

Traditionally, sample supply to the inspection cuvette of a flowcytometer has been accomplished by pressurizing a tube containing asample to be analyzed, causing the sample to flow into the sample supplyline. Automation of this traditional supply method is difficult due tomechanical issues with automatic positioning and sealing of samplecontainers. Such a supply method is also prone to creating aerosols ofbiohazardous materials.

Another method of sample supply, which is sometimes implemented in flowcytometers, is aspiration of sample into the flow cytometer syringe pumpand then expulsion of the sample into flow cytometer cuvette. Onedrawback of this method is that pulsations created in the sample flowcan greatly amplify noise on the measured signal. Additionally, theamount of time required for washing between samples is great and slowsthe process for analyzing samples. The washing process cannot beginuntil sample has been fully dispensed into the flow cytometer cuvette,resulting in reduced throughput.

Another method of sample supply is described in U.S. Pat. No. 5,182,617,which is herein incorporated by reference in its entirety. The '617patent discloses that higher throughput may be achieved by creating twoidentical branches that can perform simultaneous sample introduction andwashing. When used, analyzed samples stick to the channels of thesystem, including the interface channels, causing major carryover issuesin subsequent sample analysis. The prior art provides no provision forthorough washing of an interface channel between the sample supplydevice and the flow cytometer. Furthermore, the prior art fails to teachany provision for rapidly washing the system with a cleaning liquid.

SUMMARY OF THE INVENTION

A method for multiplexed sample analysis is provided comprising thesteps of (a) robotically obtaining a first liquid sample from the firstof a plurality of sample sources; (b) delivering the first liquid samplethrough a first line to an instrument for analysis of the sample (c)robotically obtaining a second liquid sample from the second of aplurality of sample sources; (d) delivering the second liquid samplethrough a second line to an instrument for analysis; (e) cleaning thesecond line while the first line is delivering sample to the instrument;and (f) cleaning the first line while the second line is deliveringsample to the instrument. In one embodiment, the method furthercomprises step (g) repeating steps (a)-(f) to obtain and delivermultiple samples to the instrument from all of the plurality of samplesources. Preferably, the instrument is a flow cytometer. In anotherembodiment, the instrument is selected from the group consisting of aspectrofluorometer, a fluorometer, an absorbance meter, and amicroscope.

In an embodiment, the first and second liquid samples that are analyzedby the inspection instrument may be independently selected from thegroup consisting of chemical compounds, antibodies, beads, live cells,or fixed cells. Any sample typically analyzed by a flow cytometer, HPLC,spectrofluorometer, fluorometer, absorbance meter, microscope, or otherhigh-throughput-instrument that receives liquid samples may be used.

In another embodiment, the method further comprises the steps ofdelivering a first reagent to the first liquid sample before the sampleis delivered to the instrument and delivering a second reagent to thesecond liquid sample before the sample is delivered to the instrument.The first and second reagents may be independently selected from thegroup consisting of chemical compounds, antibodies, beads, live cells,or fixed cells. In still another embodiment, multiple reagents may beadded to the sample before the sample is injected into the instrument.

In another embodiment, a control valve alternates delivery of cleaningfluid with the delivery of liquid sample in each of the first and secondlines. The control valve may be connected to additional lines. Suchlines may further provide the delivery of air pressure or system liquidpressure. Additionally, such lines may further provide a waste channel.

An apparatus for delivering samples to an instrument for analysis isprovided comprising a first and a second sample delivery line, whereinthe first sample delivery line comprises a first sample loading andinjection branch and the second sample delivery line comprises a secondsample loading and injection branch; a fluid flow management mechanismfor alternately connecting the first and second sample delivery lines tothe instrument; at least one cleaning fluid delivery line to supplycleaning fluid to the flow management mechanism, the first sampledelivery line, and the second sample delivery line; at least one sampleinput channel; at least one control valve for alternately deliveringcleaning fluid to the first and second sample delivery lines, so thatsample and cleaning fluid alternately flow through the first and secondsample delivery lines. In one embodiment, the at least one cleaningfluid delivery line supplies cleaning fluid to the entire system,including the inspection instrument.

In another embodiment, the fluid flow management mechanism comprises aposition switching valve. The position switching valve may comprise afour-way two-position switching valve. The two-position switching valvemay comprise two modes. In one embodiment, the first mode of theposition switching valve fluidicly couples the first sample deliveryline to the instrument and fluidicly couples the second sample deliveryline to the sample input channel. In another embodiment, the second modeof the position switching valve fluidicly couples the first sampledelivery line to the sample input channel and fluidicly couples thesecond sample delivery line to the instrument.

In one embodiment, the sample loading and injection branches of thefirst and second sample delivery lines comprise symmetrical sampleloading and injection branches. In another embodiment, the first sampledelivery line comprises a first sample holding loop and the secondsample delivery line comprises a second sample holding loop. In anotherembodiment, a first control valve is located within the first sampledelivery line and a second control valve is located within the secondsample delivery line. In some embodiments, the first and second controlvalves each comprise four fluidic channels capable of coupling the firstand second sample holding loops to one of the four fluidic channels.These additional fluidic channels may be connected to output ports ofpump flow control valves, at least one cleaning fluid delivery line,waste disposal, or a controlled air pressure source.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a configuration of one embodiment of thepresent invention.

FIGS. 2A and 2B show a top level diagram showing an embodiment of oneoperation of the control unit.

FIG. 3 is a diagram showing an embodiment of one sequence of controlunit commands for washing both branches of the system.

FIG. 4 is a diagram showing an embodiment of one sequence of controlunit commands for adding a reagent to sample containers on both branchesof the system.

FIG. 5 is a diagram showing an embodiment of one sequence of controlunit commands for mixing and loading samples on both branches of thesystem; and

FIGS. 6A and 6B show a diagram showing an embodiment of one sequence ofcontrol unit commands for injecting sample and washing interface line onboth branches of the system.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The term “pressurizing means” as used herein refers to a variety ofmethods for providing pressure into the channels of the device.Non-limiting examples of pressurizing means include regulated compressedair from a tank, a regulated air compressor, and a syringe pump.

The term “storage means” as used herein refers to a variety of samplecontainers from which sample is aspirated into the sample supplychannel. Non-limiting examples of storage means include a 96, 384, or1536 well plate or a rack comprising any number of test tubes.

The term “channel switching means” as used herein refers to a switchingvalve that directs fluid in the channels to desired locations in thedevice of any embodiment disclosed herein.

FIG. 1 shows one embodiment of the present invention and is intended tobe non-limiting in its scope. In FIG. 1, an inspection instrument (inthis case a flow cytometer) 1 is in fluid communication with a four-waytwo position switching valve 3 via a flexible fluidic interface channel2. The interface channel may be constructed of any known tubing materialsuitable for use with an inspection instrument. Non-limiting examples oftubing material for the interface channel and all other tubing materialsinvolved in the present invention include tubing made from flexiblesilicon, polyvinyl chlorine (PVC), polyether ether ketone (PEEK),polytetrafluoroethylene (PTFE), and any other useful polymericmaterials, such as polyesters, polyolefins, or polyamides. The interfacechannel 2 may have an internal diameter from roughly about 0.005″ toabout 0.040″, although other sizes of internal diameter may beappropriate depending on the inspection instrument used. The valve 3switches sample input channel (“input channel”) 4 and interface channel2 between two symmetrical sample loading and injection branches of thesystem 5 and 6. In a first position of the switching valve 3, branch 5is fluidicly coupled to input channel 4 and interface channel 2 (andthus, the inspection instrument 1) is fluidicly coupled to branch 6.When the valve 3 is switched into a second position (not shown), branch5 would be fluidicly coupled to interface channel 2 (and thus, theinspection instrument 1) and input channel 4 would be fluidicly coupledto branch 6.

The other end of input channel 4 and is coupled to a probe 7, which ismechanically coupled to a positioning device 8. In one embodiment, theinput channel comprises the probe and positioning device. The probe 7provides the input channel 4 with an appropriate sample. Any type ofsample that is analyzed with by an inspection instrument may be used inthe present invention, including liquid, gaseous, or solid samples.Non-limiting examples of useful samples include chemical compounds andbiological compounds. Some non-limiting examples of chemical compoundsinclude chemical reagents, solvents, microspheres, beads, and dyes.Preferably, the chemical compounds comprise compounds that are known andused in an HPLC or a flow cytometer inspection instrument. Somenon-limiting examples of biological compounds include blood, urine,antibodies, live cells, dead cells, and microorganisms. The cells may befrom humans, animals, insects, bacteria, yeast, or viruses. Preferably,the biological compounds comprise compounds that are known and used in aflow cytometer inspection instrument.

The positioning device 8 may be any device known to one of ordinaryskill in the art. The positioning device 8 may be commanded to move theprobe 7. Non-limiting examples of positioning devices include aCartesian robotic sampler and a rotary sampler. Another example of apositioning device 8 includes an XYZ positioning device, such as a TecanMSP9250 robotic sampler with travel range of 15.4″ on X axis, 11.8″ on Yaxis, and 6.5″ on Z axis. The positioning accuracy of this device is0.004″ on all axes. The Z axis of positioning device 8 is capable ofmounting probes of diameters up to 0.078″ via a set screw. In anotherembodiment, the robotic sampler comprises separate arms for delivering avariety of reagents to the samples. In another embodiment, the roboticsampler comprises separate arms, for example, one, two, three, or fourarms, for delivering a variety of samples to the inspection instrument.In another embodiment, the at least one sample input channel is coupledto a robotic sampler positioning device. In another embodiment, therobotic sampler comprises two arms and the second arm comprises areagent transfer pump. In one embodiment, the second arm delivers avariety of reagents, for example, first and second reagents. In anotherembodiment, the reagent transfer pump comprises a syringe pump, aperistaltic pump, or a diaphragm pump.

Probe 7 may be any known tubing systems useful in the transportation ofsamples within the system. In one embodiment, probe 7 comprises astainless steel tubing of about 0.072″ outer diameter (OD), and about0.063″ inner diameter (ID), although other tubing dimensions arecontemplated. For example, the OD of the probe may range between about0.050″ and 0.100″ and ID of the probe may range between about 0.045″ andabout 0.095″. A typical probe tubing length is about 4″ but could belonger or shorter depending on the type of sample containers 9 andreagent vials 11 that are used. Preferably, probe 7 is long enough to beable to reach the bottom of sample containers 9 and reagent vials 11without end of Z axis impacting top of the container. An additional 1″may be added to this minimum probe length to allow for mounting of probe7 in the end of Z axis of positioning device 8.

In one embodiment, probe 7 has a rectangular opening machined into itsside, starting about 1″ from the end of probe 7 where it is mounted ontothe Z axis of positioning device 8 and extending for about 0.5″ fromthat point. The depth of this opening may be about one half of the outerdiameter of the probe 7. Input channel 4 may pass through this openingand out of the tip of probe 7. The outer diameter of input channel 4 maybe sized so that it snugly fits within the inner diameter of the probe7. For example, the OD of input channel 4 may be about 0.0625″ when theID of the probe is 0.063″ and the input channel 4 may optionally befriction fit into probe 7. Input channel 4 may be extended from the tipof probe 7 by approximately 0.5″ to avoid contact between the outer wallof probe 7 and the contents of sample containers 9 where the samplecontainers are relatively flat, for example, a 96-well plate, a 385-wellplate, or a 1536-well plates.

The positioning device 8 can be commanded to move probe 7 among severaldevices, including sample containers 9, a wash station 10, and any ofthe reagent vials 11 located in a reagent rack 12. Many different samplecontainers 9 may be employed in the embodiments described herein.Non-limiting examples of sample containers include tube racks comprisingvarying amounts of sample tubes and well plates of 96, 384, or 1536wells.

In one embodiment, the wash station 10 is coupled to a waste reservoir13 via a large bore drain channel 14. In another embodiment, the wastereservoir 13 is physically located below the wash station 10 to allowany liquids expelled into the wash station 10 to flow into the wastereservoir 13 under gravity. The waste station drain channel should havean internal diameter large enough to prevent airlock in the line upondrainage of any waste material. The typical internal diameter of a wastestation drain channel 14 is not less than about ⅜ inches.

In an embodiment, both sample loading and injection branches 5 and 6 ofthe system are identical in configuration and are each independentlycontrolled. In an embodiment, each branch 5 and 6 consists of a sampleholding loop 15 and 16, respectively, control valves 17 and 18,respectively, channels 19 through 22 and 23 through 26, respectively,pump flow control valves 27 and 28, respectively, and pumps 29 and 30,respectively. In one embodiment, the sample holding loops 15 and 16 arefluidicly coupled to the switching valve 3. In an embodiment, the sampleholding loops 15 and 16 are constructed of a length of tubing havinginternal volume greater than the maximum volume of sample that can beloaded and injected by the system. This ensures that the sample holdingloops 15 and 16 have sufficient volume to contain any sample that may beinjected therein.

The other end of each holding loop 15 and 16 is fluidicly coupled tocontrol valves 17 and 18 respectively. Typically, holding loops 15 and16 would have internal volume one and one half times greater than themaximum volume of sample to prevent sample from being aspirated intocontrol valves 17 and 18. In one embodiment, both control valves 17 and18 are selection valves comprising four channels capable of couplingsample holding loops 15 and 16 to one of the four fluidic channels. Forembodiments not requiring as many fluidic channels, the selection valvesmay comprise as few as two channels. In other embodiments, selectionvalves with more than four channels can be used in order to provideadditional washing capabilities of the system.

Control valves 17 and 18 having multiple channels provide manyadvantageous features for the invention described herein. In FIG. 1,control valves 17 and 18 selectively connect sample holding loops 15 and16 to one of four possible channels. Holding loop 15 is connected tochannels 19, 20, 21, and 22 and holding loop 16 is connected to channels23, 24, 25, and 26.

In an embodiment, the control valves 17 and 18 comprise channels forpumps. Pump channels 19 and 23 are connected to output ports of pumpflow control valves 27 and 28 respectively. Pump flow control valves 27and 28 are normal three way valves with a common port, a normally openport, and a normally closed port. The common ports of pump flow controlvalves 27 and 28 are connected to pumps 29 and 30, which are capable ofaspirating and dispensing liquids. In an embodiment, pumps 29 and 30 areeach independently a syringe pump. However, other types of pumps may beused. Non-limiting examples include peristaltic pumps and diaphragmpumps. In an embodiment, the input ports of pump flow control valves 27and 28 are connected to a system liquid reservoir 31. Any system liquiduseful in allowing or aiding performance of the inspection instrumentmay be used. Non-limiting examples include water, saline, orphosphate-buffered saline.

In an embodiment, the control valves 17 and 18 comprise channels forholding cleaning fluid to wash and clean the system. Wash channels 20and 24 are both fluidicly coupled to each other and a wash pump 32 attee junction 33. The wash pump 32 is fluidicly coupled to a common portof a three way wash liquid selection valve 34 that comprises a commonport, a normally open port, and a normally closed port. The normallyopen port of valve 34 is connected to system liquid reservoir 31 viachannel 35 and the normally closed port of valve 34 is connected tocleaning liquid reservoir 37 via channel 36. Wash pump 32 in thisembodiment is a diaphragm pump capable of rapidly pumping cleaningliquids to control valves 17 and 18. Other non-limiting examples ofsuitable pumps that can be used for pumping liquids through the systeminclude peristaltic, syringe, and a pressurized reservoir.

In an embodiment, the control valves 17 and 18 comprise channels forwaste. Waste channels 21 and 25 are both fluidicly coupled to each otherand a waste reservoir 13 at a tee junction 38.

In an embodiment, the control valves 17 and 18 comprise channels forcontrolling the pressure in the channels. Pressurized channels 22 and 26are both fluidicly coupled to each other and a pressurized system liquidreservoir 39 at a tee junction 40. The pressurized system liquidreservoir 39 is pressurized via a carefully controlled air pressuresource 41. Controlled air pressure sources useful in the presentinvention include a regulated compressed air tank or a regulated aircompressor. Alternatively, the pressure source may come from theinspection instrument 1 if such source is available. However,pressurized channels 22 and 26 are preferred when very precisemeasurements are to be taken and injecting samples with pumps 29 and 30would increase the signal to noise ratio.

One improvement provided by the control valves 17 and 18 is thecapability to rapidly wash lines 15 and 16 with a cleaning liquid 37. Asticky compound is any compound that remains in the lines of the systemthat has the potential to contaminate future samples. Often, stickycompounds, for example compounds such as sphingosine-1-phosphate (SIP),endothelin-1 (ET-1), and rhodamine, are involved when using inspectioninstruments, such as flow cytometers. It is therefore essential to cleansystem lines between sample inspections with a cleaning solution inorder to eliminate any carryover that may exist from previous samples.Typical system liquids, such as saline solution, as compared to cleaningliquids, are prone to leave behind sticky compounds in the lines of thesystem. This causes contamination for hundreds of samples that areanalyzed afterward.

Another feature of the control valves 17 and 18 is that they allow theinspection instrument 1 to back-flush remaining sample in interface line2 back through the apparatus into waste. As sample travels throughinterface line 2 and lines 15 and 16, it becomes longer because samplein the center of the tube travels faster than at the surface. This oftenleads to a situation where the whole sample is never fully injected intothe inspection instrument.

This may be accomplished by connecting line 2 to one of the lines 15 and16 and also connecting control valves 17 and 18 to waste channels 21 and25. Where a flow cytometer is used as the inspection instrument, it maypressurize the input line 2, making the connection to a waste line thatis open to atmospheric pressure. This often causes a rapid flow of cleanliquid from inspection instrument 1 through interface line 2. Thisfeature makes it possible to carry out inspection of samples at highspeed because it overcomes the problem of waiting for extended periodsof time for sample to clear the tubing.

Another feature of the control valves 17 and 18 is that they allow thecapability to load large sample volumes utilizing syringes 29 and 30.The large sample volumes may then be injected with high precisionutilizing pressurized system liquid 39. This is particularly importantas syringe size increases. As syringe size increases, the minimum flowrate that a syringe pump can sustain without fluctuations alsoincreases. As the flow rate increases, the inspection instrument(particularly a flow cytometer) loses precision in measuring thesamples. The present invention described herein allows one of ordinaryskill in the art to load a large sample, such as 1 mL, 2 mL, or evengreater, and then inject it very slowly allowing for the same precisionfound in manual tube injection, but with the additional advantage offull automation.

In another embodiment, the pressurized system liquid vessel 39 may bereplaced with a high-resolution syringe pump. High-resolution syringepumps are capable of supplying larger volumes of liquid at very low flowrate. Inserting a high-resolution syringe pump may be accomplished byreplacement of the pressurized system liquid vessel 39.

The invention described herein provides the capability to deliver one ormore reagents to sample containers before they are injected into thesystem by utilizing a separate auto-sampler arm comprising its ownsyringe for aspirating or dispensing. Such a feature would preventcarryover from sample containers back to the reagent if the auto-samplerarm carrying a reagent dispenses that reagent into the wells above theliquid surface of the sample. In an embodiment, the auto-samplercomprises multiple arms, such as an auto-sampler comprising two, three,or four arms. In another embodiment, the auto-sampler comprises twoarms. Where cells are utilized as the reagent and a single sameauto-sampler arm is used to take cells to the sample, mix and inject,and then go back to cells, it is possible that carryover of unwashedparts of the sample back to the cells may occur. One example wheremultiple reagents may be added to the sample before injection in theinstrument comprises having agonist compounds on a plate well and addingcells to the well before they are injected into the inspectioninstrument. Other combinations of reagents and samples, as describedherein, may be substituted for agonists and cells. Another examplecomprises placing an allosteric modulator or other antagonist compoundsin a plate well, adding cells from a reagent vial, and then finallyadding another agonist compound into the well. The mixture may then beinjected into the flow cytometer. Each sample may have different typesand varying numbers of reagents added thereto. In one embodiment,delivery of first and second reagents, and optionally additionalreagents is performed with a separate auto-sampler arms.

In some embodiments, the channels comprising the system are made withpolymer tubing having internal diameter of about 0.02 inches. In anotherembodiment, a PFA (PerFluoroAlkoxy) material is used to minimize anyissues associated with gas permeability. Channels connected to thesystem liquid reservoir 31 are typically made with tubing having largerinternal diameter such as about 1/16 inches or about 0.094 inches. Inanother embodiment, the holding loops 15 and 16 are made out of largerinternal diameter tubing if switching valve 3 and control valves 17 and18 are in close physical proximity in order to reduce the requiredlength of tubing.

In an embodiment, a control unit 42 is electrically connected toswitching valve 3, positioning device 8, wash pump 32, wash liquidselection valve 34, control valves 17 and 18, pump flow control valves27 and 28, and pumps 29 and 30. The control unit 42 is implemented as acomputer capable of independent control of all attached devices. Controlunit 42 may also set the air supply pressure 41 via an adjustableregulator. The air supply pressure can also be controlled via a manualpressure regulator. In an embodiment, the air supply pressure 41 is setto a value slightly higher than backpressure generated by inspectioninstrument 1. For example, the air supply pressure may be set to betweenabout 0.5 and about 2.0 psi over the backpressure.

The operation of the foregoing apparatus having the above-describedconfiguration will now be explained in some non-limiting embodiments.The control of the operation is preferably performed according tocommands from control unit 42.

Initially, an operation is performed to fill all system channels (exceptfor waste channels 21 and 25) with system liquid. Positioning device 8is commanded to move the probe 7 into the wash station 10. The pump flowcontrol valve 27 is switched to communicate system liquid from reservoir31 to pump 29. Thereafter, pump 29 is filled with system liquid bycommand from control unit 42. The pump flow control valve 27 is thenswitched to communicate system liquid in pump 29 to pump channel 19. Thevalve 3 is switched into position to communicate liquid between inputchannel 4 and holding loop 15.

Next, control valve 17 is switched to communicate liquid between holdingloop 15 and pump channel 19. The pump 29 is commanded to completelyexpel system liquid into pump channel 19 and holding loop 15 effectivelypurging air and filling both with the system liquid. Valve 17 is thenswitched to communicate liquid between holding loop 15 and wash channel20, which connects to the wash pump 32. The wash liquid selection valve34 is switched to fluidicly couple the wash pump 32 and cleaning liquidreservoir 37 via cleaning liquid channel 36. Wash pump 32 is then turnedon to fill cleaning liquid channel 36 with a cleaning liquid. Anycleaning liquid capable of removing sample residue and providing anon-contaminated surface for the channels may be selected. Somenon-limiting examples of cleaning liquids include solvents such asethanol, dimethyl sulfoxide (DMSO), or a detergent. Wash liquidselection valve 34 is then switched to fluidicly couple wash pump 32 andsystem liquid reservoir 31 via channel 35. The system liquid fills washpump 32, wash channel 20, control valve 17, holding loop 15, switchingvalve 3, input channel 4 and probe 7 with system liquid.

The wash pump 32 is then turned off after a fixed delay sufficient tofill components mentioned above with the system liquid. If pressurizedsystem liquid reservoir 39 is connected to control valve 17, then valve17 is switched to communicate the pressurized system liquid fromreservoir 39 to holding loop 15 via pressurized channel 22. After adelay to fill pressurized channel 22 with system liquid, control valve17 is switched to communicate liquid in holding loop 15 to the wastechannel 21.

In order to fill the second branch of the system with system liquid,valve 3 is switched into position to communicate liquid between inputchannel 4 and holding loop 16. The sequence of events described abovefor the first branch of the system is then repeated for the equivalentparts of the second branch of the system. The resulting state of thesystem is with probe 7 in wash station 10 and all channels of the system(except waste channels 21 and 25) filled with system liquid. In anotherembodiment, the process of filling lines with system liquids may furtherbe practiced with additional branches.

FIGS. 2A and 2B show another embodiment of the present invention and areintended to be non-limiting in its scope. FIGS. 2A and 2B show asequence of steps to evaluate one or more samples from sample containers9. In step 43, which is the starting point of FIG. 2A, a wash cycle 68(further described in FIG. 3) is executed on branch 5. Wash cycle 68ensures that branch 5 has been washed and is filled with system liquid.In some embodiments, it may be beneficial to add a reagent to samplecontainers 9 before the sample is analyzed. Examples of non-limitingreagents include agonists, antagonists, modulators, dyes, stains, cells,and beads. Where a user selects to add a reagent to the sample in step44, a reagent addition cycle 94 is executed on branch 5 in step 45. Instep 46, the first sample is loaded into holding loop 15 by executingmix and load next sample cycle 110 (further described in FIG. 5) onbranch 5. In step 47, switching valve 3 is switched to fluidicly coupleholding loop 15 and interface channel 2. Two concurrent and independentprocesses are then executed. The first process comprises step 48 and thesecond process comprises steps 49 through 53.

In the first process, a sample is injected into the inspectioninstrument 1 and the interface channel 2 is washed in step 48 by runninginject sample and wash interface line cycle 128 (further described inFIG. 6A) on branch 5. The second process first determines if there areany more samples left to be processed in step 49. If all of the sampleshave been processed, the second process terminates by going to step 54.Otherwise branch 6 is washed in step 50 by executing a wash cycle 81(further described in FIG. 3). If the user selected to add a reagent tothe sample in step 51, a reagent addition cycle 102 (further describedin FIG. 4) is executed on branch 6 in step 52. In step 53, the nextsample is loaded into holding loop 16 by executing mix and load nextsample cycle 119 (further described in FIG. 5) on branch 6. Uponcompletion of both processes in step 54, a check is made in step 55,shown in FIG. 2B, to determine if all samples have been processed. If nomore samples remain, then the execution continues at step 65.

If there are still samples to be processed, switching valve 3 isswitched to fluidicly couple holding loop 16 and interface channel 2 instep 56. Two concurrent and independent processes are then executed. Thefirst process comprises step 57 and the second process comprises steps58 through 62.

The first process injects sample into inspection instrument 1 and washesinterface channel 2 in step 57 by running inject sample and washinterface line cycle 137 (further described in FIG. 6B) on branch 6. Thesecond process first determines if there are any more samples left to beprocessed in step 58. If all samples have been processed, then thesecond process terminates by going to step 63. Otherwise branch 5 iswashed in step 59 by executing a wash cycle 68. If the user selected toadd a reagent to the sample in step 60, a reagent addition cycle 94 isexecuted on branch 5 in step 61. In step 62, the next sample is loadedinto holding loop 15 by executing mix and load next sample cycle 110(further described in FIG. 5) on branch 5. Upon completion of bothprocesses in step 63 a check is made in step 64 to determine if allsamples have been processed. If no more samples remain the executioncontinues at step 65, otherwise execution continues at step 47 describedabove.

In step 65, branch 5 is washed by executing wash cycle 68. Then, branch6 is washed by executing wash cycle 81 in step 66. Sample processingcompletes in step 67 with the system in a ready state to startprocessing another set of samples upon replacement of sample containers9.

FIG. 3 shows another embodiment of the present invention and is intendedto be non-limiting in its scope. FIG. 3 shows a sequence of stepswherein both branches of the system described herein are washed. Theinitial wash cycle 68 begins by moving probe 7 to wash station 10 instep 69. In step 70, control valve 17 is switched to connect washchannel 20 to holding loop 15. In some embodiments, samples to beintroduced into the inspection instrument 1 may be hydrophobic and proneto sticking to the channel walls. It is often necessary to wash thechannels with a cleaning liquid to reduce chances of cross contaminationfrom one sample to the next. Any cleaning liquid capable of removingsample residue and providing a non-contaminated surface for the channelsmay be selected. Some non-limiting examples of cleaning liquids includesolvents such as ethanol, dimethyl sulfoxide (DMSO), or a detergent.

In step 71, the wash liquid selection valve 34 is switched to connectcleaning liquid channel 36 and wash pump 32. The wash pump 32 is thenturned on in step 72 to pump cleaning fluids into the control valves. Auser specified delay of X1 seconds is introduced at step 73 to allowcleaning liquid to be pumped all the way through valve 17, holding loop15, switching valve 3, input channel 4 and probe 7 into wash station 10.The actual amount of time needed may vary depending on the system beingused and the length of the channels. One of ordinary skill in the art,guided by the disclosure provided herein, can determine how much timewill be required for the cleaning liquids to be pumped throughout thesystem.

With the wash pump 32 still running, the wash liquid selection valve 34is switched to connect channel 35 to the wash pump 32, as shown in step74. This action establishes a flow of system liquid from system liquidreservoir 31 through the wash pump 32. A user specified delay of Y1seconds is introduced at step 75 to allow system liquid to be pumped allthe way through valve 17, holding loop 15, switching valve 3, inputchannel 4 and probe 7 into wash station 10, thus washing out allremaining cleaning liquid. The actual amount of time needed may varydepending on the system being used and the length of the channels. Oneof ordinary skill in the art, guided by the disclosure provided herein,can determine how much time will be required to wash out the cleaningliquids.

Wash pump 32 is turned off in step 76 and control valve 17 is switchedto connect pump channel 19 and holding loop 15 in step 77. In step 78,pump flow control valve 27 is switched to connect pump channel 19 andpump 29. All fluid is then expelled out of pump 29 in step 79. Step 80shows the completion of wash cycle 68.

The wash cycle 81 for branch 6 of the system is the same as wash cycle68 for branch 5 with all elements of branch 5 replaced withcorresponding elements of branch 6. The wash cycle 81 is illustrated insteps 82 through 93. The initial wash cycle 81 begins by moving probe 7to wash station 10 in step 82. In step 83, control valve 18 is switchedto connect wash channel 24 to holding loop 16. As previously stated inregards to wash cycle 68, some embodiments involve samples introducedinto the inspection instrument 1 that are hydrophobic and prone tosticking to the channel walls. Similar cleaning liquids may be used inregards to wash cycle 81 that are useful in regards to wash cycle 68.

In step 84, the wash liquid selection valve 34 is switched to connectcleaning liquid channel 36 and wash pump 32. The wash pump 32 is thenturned on in step 85 to pump cleaning fluids into the control valves. Auser specified delay of X2 seconds is introduced at step 86 to allowcleaning liquid to be pumped all the way through valve 18, holding loop16, switching valve 3, input channel 4 and probe 7 into wash station 10.The amount of X2 seconds used in step 86 may be the same or differentfrom the amount of time used in step 73 for wash cycle 68 on branch 5.One of ordinary skill in the art, guided by the disclosure providedherein, can determine how much time will be required for the cleaningliquids to be pumped through the system.

With the wash pump 32 still running, the wash liquid selection valve 34is switched to connect channel 35 to the wash pump 32, as shown in step87. This action establishes a flow of system liquid from system liquidreservoir 31 through the wash pump 32. A user specified delay of Y2seconds is introduced at step 88 to allow system liquid to be pumped allthe way through valve 18, holding loop 16, switching valve 3, inputchannel 4 and probe 7 into wash station 10, removing all of theremaining cleaning liquid. The actual amount of time needed may varydepending on the system being used and the length of the channels. Theamount of Y2 seconds used in step 88 may be the same or different fromthe amount of time used in step 75 for wash cycle 68 on branch 5. One ofordinary skill in the art, guided by the disclosure provided herein, candetermine how much time will be required to wash out the cleaningliquids.

Wash pump 32 is turned off in step 89 and control valve 18 is switchedto connect pump channel 23 and holding loop 16 in step 90. In step 91,pump flow control valve 28 is switched to connect pump channel 23 andpump 30. All fluid is then expelled out of pump 30 in step 92. Step 93shows the completion of wash cycle 81.

FIG. 4 shows another embodiment of the present invention and is intendedto be non-limiting in its scope. In FIG. 4, a sequence of steps to addreagent on both branches of the system is described. Reagent additioncycle 94 starts by switching control valve 17 to connect holding loop 15and pump channel 19 in step 95. Then in step 96, pump flow control valve27 is switched to connect pump channel 19 and pump 29. The probe 7 isthen moved to a user selected reagent vial 11 in step 97. A volume ofreagent is aspirated into probe 7 and input channel 4 by issuing anaspirate command to pump 29 in step 98. The volume of reagent aspiratedinto the probe and probe 7 and input channel 4 is selected by the userof the system and may be any amount necessary to complete the relevanttests.

The probe 7 is then moved from the reagent vial into the samplecontainer of the next sample to be loaded in step 99. The reagent isthen dispensed into sample from input channel 4 by issuing a dispensecommand to pump 29 in step 100. Reagent addition cycle 94 is shown ascompleted in step 101.

Reagent addition cycle 102 for branch 6 of the system is the same asreagent addition cycle 94 for branch 5 with all elements of branch 5replaced with corresponding elements of branch 6. The reagent additioncycle 102 is illustrated in steps 103 through 109. Reagent additioncycle 102 starts by switching control valve 18 to connect holding loop16 and pump channel 23 in step 103. Then in step 104, pump flow controlvalve 28 is switched to connect pump channel 23 and pump 30. The probe 7is then moved to a user selected reagent vial 11 in step 105. A volumeof reagent is aspirated into probe 7 and input channel 4 by issuing anaspirate command to pump 30 in step 106. The volume of reagent aspiratedinto the probe and probe 7 and input channel 4 is selected by the userof the system and may be any amount necessary to complete the relevanttests.

The probe 7 is then moved from the reagent vial into the samplecontainer of the next sample to be loaded in step 107. The reagent isthen dispensed into sample from input channel 4 by issuing a dispensecommand to pump 30 in step 108. Reagent addition cycle 102 is shown ascompleted in step 109.

FIG. 5 shows another embodiment of the present invention and is intendedto be non-limiting in its scope. In FIG. 5, a sequence of steps to mixand load the next sample on both branches of the system is described.For some types of samples, such as living cells or beads that may settleat the bottom of sample containers 9, it may be desired to mix thesample several times before aspirating it into holding loop 70. Mix andload next sample cycle 110 begins by switching control valve 17 toconnect holding loop 15 and pump channel 19 in step 111. In step 112,pump flow control valve 27 is switched to connect pump channel 19 andpump 29. The probe 7 is then moved into a sample container 9 of the nextsample to be loaded in step 113. A user defined sample mix volume isaspirated into the probe 7 and input channel 4 by issuing an aspiratecommand to pump 29 in step 114. The sample is then dispensed back intosample container 9 by issuing a dispense command to pump 29 in step 115.

The system user is able to define any number of mixing cycles necessaryto complete the desired testing. If the user defined number of mixingcycles has not been completed in step 116 then execution repeats fromstep 114. Otherwise, a user defined sample volume and input channel deadvolume is aspirated into holding loop 15 with pump 29 in step 117. Themix and load next sample cycle 110 is shown as completed in step 118.

Mix and load next sample cycle 119 for branch 6 of the system is thesame as mix and load next sample cycle 110 for branch 5 with allelements of branch 5 replaced with corresponding elements of branch 6.The mix and load next sample cycle 119 is illustrated in steps 120through 127. Mix and load next sample cycle 119 begins by switchingcontrol valve 18 to connect holding loop 16 and pump channel 23 in step120. In step 121, pump flow control valve 28 is switched to connect pumpchannel 23 and pump 30. The probe 7 is then moved into a samplecontainer 9 of the next sample to be loaded in step 122. A user definedsample mix volume is aspirated into the probe 7 and input channel 4 byissuing an aspirate command to pump 30 in step 123. The sample is thendispensed back into sample container 9 by issuing a dispense command topump 30 in step 124.

The system user is able to define any number of mixing cycles necessaryto complete the desired testing. If the user defined number of mixingcycles has not been completed in step 125 then execution repeats fromstep 123. Otherwise, a user defined sample volume and input channel deadvolume is aspirated into holding loop 16 with pump 30 in step 126. Themix and load next sample cycle 119 is shown as completed in step 127.

FIGS. 6A and 6B show another embodiment of the present invention and areintended to be non-limiting in its scope. In FIG. 6A, a sequence ofsteps to inject the sample and wash interface line 2 on both branches ofthe system is described. The inject sample and wash interface line cyclebegins at step 128. After a sample is injected into the inspectioninstrument 1, the time in which it takes the sample to traverseinterface channel 2 may be significant. This is especially true if theinspection instrument 1 is a flow cytometer, where rates can be as slowas approximately 1 μL/sec. To reduce time for sample injection, a userdefined boost volume corresponding to internal volume of interfacechannel 2 is expelled from the holding loop 15 into inspectioninstrument 1 at a high speed in step 129. For example, the rate at whichthe sample is injected into the inspection instrument may be as high asabout 10 μL/sec or greater. In another embodiment, the rate at which thesample is injected into the inspection instrument may be as high asabout 50 μL/sec or greater. In still another embodiment, the rate atwhich the sample is injected into the inspection instrument may be ashigh as about 100 μL/sec or greater. This effectively brings the frontof the sample plug into the inspection instrument 1 in a short amount oftime.

The injection may be made either with pressure or without pressure. Instep 130, if a user did not select to use a pressurized sample delivery,then the sample is injected into inspection instrument 1 by dispensing auser defined sample volume with pump 29 at normal speed in step 131.Otherwise steps 132 and 133 are executed. In step 132, control valve 17is switched to connect holding loop 15 and pressurized channel 22. Theuser may specify a delay sufficient to inject the sample into theinspection instrument in step 133. In step 134, control valve 17 isswitched to connect holding loop 15 and waste channel 21. The user mayspecify a cleaning delay in step 135 to allow back pressure frominspection instrument 1 to backwash interface channel 2 into the wastereservoir 13. Inject sample and wash interface line cycle 128 iscompleted as shown in step 136.

FIG. 6B shows inject sample and wash interface line cycle 137 for branch6 of the system and is the same as inject sample and wash interface linecycle 128 for branch 5 with all elements of branch 5 replaced withcorresponding elements of branch 6. The inject sample and wash interfaceline cycle 137 is illustrated in steps 138 through 145. A user definedboost volume corresponding to internal volume of interface channel 2 isexpelled from the holding loop 16 into inspection instrument 1 at a highspeed in step 138. For example, the rate at which the sample is injectedinto the inspection instrument may be as high as about 10 μL/sec orgreater. In another embodiment, the rate at which the sample is injectedinto the inspection instrument may be as high as about 50 μL/sec orgreater. In still another embodiment, the rate at which the sample isinjected into the inspection instrument may be as high as about 100μL/sec or greater. This effectively brings the front of the sample pluginto the inspection instrument 1 in a short amount of time.

The injection may be made either with pressure or without pressure. Instep 139, if a user did not select to use a pressurized sample delivery,then the sample is injected into inspection instrument 1 by dispensing auser defined sample volume with pump 30 at normal speed in step 140.Otherwise steps 141 and 142 are executed. In step 141, control valve 18is switched to connect holding loop 16 and pressurized channel 26. Theuser may specify a delay sufficient to inject the sample into theinspection instrument in step 142. In step 143, control valve 18 isswitched to connect holding loop 16 and waste channel 25. The user mayspecify a cleaning delay in step 144 to allow back pressure frominspection instrument 1 to backwash interface channel 2 into the wastereservoir 13. Inject sample and wash interface line cycle 137 iscompleted as shown in step 145.

The high-speed sample supply device disclosed herein is useful todeliver samples to any type of inspection instrument. Preferably, thedevice disclosed herein is used to delivery samples to a flow cytometer.Flow cytometers are well-known analytical tools that are able to analyzeseveral thousand particles every second and can actively separate andisolate particles having specified properties. For example, thehigh-speed sample supply device may be used in flow cytometers describedin U.S. Pat. Nos. 6,713,019; 5,824,269; 5,367,474; 5,135,502; and4,702,598; all of which are hereby incorporated by reference in theirentirety.

All patents incorporated by reference herein are incorporated byreference herein only with respect to the particular embodiments,materials, processes of manufacture and methods of use describedtherein. These patent are not to be considered incorporated by referenceto the extent any of these patents expresses an opinion or presents anyrepresentation, characterization, or definition (either expressly or byimplication) that is inconsistent with the opinions, representations,characterizations or definitions expressly made herein.

While there have been described herein what are to be consideredexemplary and preferred embodiments of the present invention, othermodifications of the invention will become apparent to those skilled inthe art from the teachings herein. It is therefore desired to be securedin the appended claims all such modifications as fall within the truespirit and scope of the invention. Accordingly, what is desired to besecured by Letters Patent is the invention as defined and differentiatedin the following claims.

1. A method for multiplexed sample analysis, comprising: (a) roboticallyobtaining a first liquid sample from the first of a plurality of samplesources; (b) delivering the first liquid sample through a first line toan instrument for analysis of the sample; (c) robotically obtaining asecond liquid sample from the second of a plurality of sample sources;(d) delivering the second liquid sample through a second line to aninstrument for analysis; (e) cleaning the second line while the firstline is delivering sample to the instrument; and (f) cleaning the firstline while the second line is delivering sample to the instrument. 2.The method of claim 1, further comprising: (g) repeating steps (a)-(f)to obtain and deliver multiple samples to the instrument from all of theplurality of sample sources.
 3. The method of claim 1, wherein theinstrument is a flow cytometer.
 4. The method of claim 1, wherein thefirst and second liquid samples are independently selected from thegroup consisting of chemical compounds, antibodies, beads, live cells,or fixed cells.
 5. The method of claim 1, further comprising the stepsof: delivering a first reagent to the first liquid sample before thesample is delivered to the instrument; and delivering a second reagentto the second liquid sample before the sample is delivered to theinstrument.
 6. The method of claim 5, wherein delivery of the first andsecond reagents is performed with a separate auto-sampler arm.
 7. Themethod of claim 5, wherein the first and second reagents areindependently selected from the group consisting of chemical compounds,antibodies, beads, live cells, or fixed cells.
 8. The method of claim 5,wherein multiple reagents are added to the sample before the sample isinjected into the instrument.
 9. The method of claim 1, wherein acontrol valve alternates delivery of cleaning fluid with the delivery ofliquid sample in each of the first and second lines.
 10. The method ofclaim 9, wherein the control valve further provides the delivery of airpressure or system liquid pressure.
 11. The method of claim 9, whereinthe control valve further provides a waste channel.
 12. An apparatus fordelivering samples to an instrument for analysis, comprising: a firstand a second sample delivery line, wherein the first sample deliveryline comprises a first sample loading and injection branch and thesecond sample delivery line comprises a second sample loading andinjection branch; a fluid flow management mechanism for alternatelyconnecting the first and second sample delivery lines to the instrument;at least one cleaning fluid delivery line to supply cleaning fluid tothe flow management mechanism, the first sample delivery line, and thesecond sample delivery line; at least one sample input channel; at leastone control valve for alternately delivering cleaning fluid to the firstand second sample delivery lines, so that sample and cleaning fluidalternately flow through the first and second sample delivery lines. 13.The apparatus of claim 12, wherein the fluid flow management mechanismcomprises a position switching valve.
 14. The apparatus of claim 13,wherein the position switching valve comprises a four-way two-positionswitching valve.
 15. The apparatus of claim 13, wherein the positionswitching valve switches between a first mode and a second mode.
 16. Theapparatus of claim 15, wherein the first mode of the position switchingvalve fluidicly couples the first sample delivery line to the instrumentand fluidicly couples the second sample delivery line to the sampleinput channel.
 17. The apparatus of claim 15, wherein the second mode ofthe position switching valve fluidicly couples the first sample deliveryline to the sample input channel and fluidicly couples the second sampledelivery line to the instrument.
 18. The apparatus of claim 12, whereinthe sample loading and injection branches of the first and second sampledelivery lines comprise symmetrical sample loading and injectionbranches.
 19. The apparatus of claim 12, wherein the first sampledelivery line comprises a first sample holding loop and the secondsample delivery line comprises a second sample holding loop.
 20. Theapparatus of claim 19, comprising a first control valve located withinthe first sample delivery line and a second control valve located withinthe second sample delivery line.
 21. The apparatus of claim 20, whereinthe first and second control valves each comprise four fluidic channelscapable of coupling the first and second sample holding loops to one ofthe four fluidic channels.
 22. The apparatus of claim 13, wherein one ofthe four fluidic channels is connected to output ports of pump flowcontrol valves.
 23. The apparatus of claim 13, wherein one of the fourfluidic channels is connected to the at least one cleaning fluiddelivery line.
 24. The apparatus of claim 13, wherein one of the fourfluidic channels is connected to waste disposal.
 25. The apparatus ofclaim 13, wherein one of the four fluidic channels is connected to acontrolled air pressure source.
 26. The apparatus of claim 12, whereinthe at least one sample input channel is coupled to a robotic samplerpositioning device.
 27. The apparatus of claim 26, wherein thepositioning device comprises one or two arms.
 28. The apparatus of claim27, wherein the positioning device comprises two arms and the second armcomprises a reagent transfer pump.
 29. The apparatus of claim 28,wherein the reagent transfer pump comprises a syringe pump, aperistaltic pump, or a diaphragm pump.
 30. The apparatus of claim 12,wherein the instrument comprises a flow cytometer.